<p><strong>In connection with increasing requirements on high throughput analyses in the laboratories the instrumental techniques undergo continuous improvement. This article is focused on the progress in the most widespread separation technique, the liquid chromatography. Its novel modification, Ultra High Performance Liquid Chromatography (UHPLC), uses the principle of separation on porous “sub-2 μm“ particles. The advantages of UHPLC over the traditional HPLC are a nine-fold reduction of analysis time, double improvement of resolution, and triple improvement of sensitivity. It results not only in time saving but also in the cost of energy and organic solvents and their ecological disposal. An alternative to UHPLC is the use of columns with core-shell particles and/or monolithic columns. The UHPLC has already been successfully applied in the analysis of beer and brewery materials.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>High performance liquid chromatography represents nowadays the most widespread separation technique applied commonly in pharmacy, toxicology, clinical analysis as well as in various research fields (Stroh et al. 2008, Pratet et al. 2004, Klein et Rivera 2004) and, last but not least, in food chemistry, namely for the analysis of beer and its raw materials (e.g. Čulík et al. 2009).</p>
<p>Increasing requirements on high throughput of samples motivate a continuous improvement of analytical techniques. During the last decade the HPLC technique has also become “faster”. There are two basic approaches to “fast chromatography”. The first one leads to the development of a completely new generation of chromatographic columns and instrumentation operating at 100 MPa (15 000 PSI) (Swartz 2005). This method was established and termed as <strong>Ultra High Performance Liquid Chromatography (UHPLC)</strong>. The second approach maintains the classical HPLC instrumentation that works at a maximal pressure of 40 MPa (6000 PSI). However, only new types of HPLC instruments comply with this pressure limitation. Analysis acceleration is achieved by the use of special columns, namely <strong>monolithic columns and columns with core-shell particles.</strong></p>
<h3><strong>2 UHPLC –ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY</strong></h3>
<p>The complex process of chromatographic separation is determined by a set of analyte interactions between mobile (solvent) and stationary phase (in simplified terms column packing). During analyte passage through the chromatographic column the analyte is carried by the mobile phase. The analyte has specific affinity to the mobile phase (it is soluble in it) and simultaneously interacts with the stationary phase (mass transfer). The contact of analyte with the stationary phase is mediated by “diffusion”. There exist three types of diffusion during the chromatographic process - longitudinal diffusion (a movement of the analyte in the mobile phase), turbulent diffusion (a movement of the analyte to the surface of the stationary phase particles and back), and diffusion inside the pores of stationary phase particles in various directions (mass transfer of analyte to the stationary phase and back). The mathematic formulation of the contribution of individual diffusion modes is called <em>van Deemter equation</em></p>
<p>H = a(dp) + b/u + c(dp)²u</p>
<p>The equation determines the relationship between the height of theoretical plate <strong>H</strong> (inverse value of column efficiency) and linear velocity <strong>u</strong> of the mobile phase. The contribution <strong>a</strong> (longitudinal diffusion) is influenced only by particle size, contribution <strong>b</strong> (turbulent diffusion) is inversely proportional to the linear velocity <strong>u</strong>, and contribution <strong>c</strong> (mass transfer) is directly proportional to a square of particle size of the stationary phase.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_1_Van_Deemterova_krivka_pro_castice_o_prumeru_10_5_3_a_1_7_mm_bc7d9c667a/Obr.-1-Van-Deemterova-krivka-pro-castice-o-prumeru-10-5-3-a-17-mm.jpg" alt="Fig. 1 Van Deemter curves for 10, 5, 3, and 1.7 μm particle size"/></p>
<p>Since the seventies, when the particles with 10 μm diameter were used, a huge progress has been achieved in the development of technologies for generation of smaller particles, which led to permanent improvement of separation parameters. Fig. 1 shows four van Deemter curves for different size particles that are commonly used (10, 5 a 3 μm and newly 1.7 μm). It follows from the figure that decrease of particle size leads to increase of separation efficiency (in other words to a decrease in plate height). This reflects the fact that the use of the smaller particle size ensures better mass transfer and simultaneously minimizes band broadening. It has impact on increased efficiency and, in addition, makes it possible to work with higher mobile phase flow and achieve thus a faster separation. This consideration led to the development of “sub-2 μm particles” (Mellors et Jorgenson 2004) providing an impetus for evolving Ultra-High Performance Liquid Chromatography (UHPLC). Using this technology, the analysis time can be reduced 9 times, sensitivity can be improved 3 times, and resolution 2 times. Such reduction of the time of analysis results not only in saving of energy and manhours but also in 10-fold reduced demand for organic solvents and in reduced costs of safe waste disposal. Evaluation of analytic parameters and comparison of general HPLC and UHPLC chromatograms is demonstrated in Fig. 2. It should be noted that columns packed with sorbent with such small particle size generate much higher back pressure than common chromatographic columns. Hence, a new UHPLC instrument had to be developed to be able to work under ultra-high pressures such as 100 Mpa (15 000 PSI). </p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_2_Porovnani_HPLC_a_UHPLC_chromatogramu_884f392190/Obr.-2-Porovnani-HPLC-a-UHPLC-chromatogramu.jpg" alt="Fig. 2 Comparison of UHPLC and HPLC chromatograms (The table lists the UHPLC characteristics superior to HPLC)"/></p>
<p>Fig. 3 demonstrates the size of new 1.7 μm particle in comparison with both a common 5 μm particle and the diameter of human hair (60 μm).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_3_Srovnani_velikosti_hybridni_1_7_mm_castice_afd22180e8/Obr.-3-Srovnani-velikosti-hybridni-17-mm-castice.jpg" alt="Fig. 3 Size comparison of 1.7 μm hybrid particle (C), common 5 μm particle (A) and diameter of a human hair 60 μm (B)"/></p>
<p>The information about the first commercially available Ultra High Performance Liquid Chromatography (UHPLC) (Schwartz 2005) system including subsequently the principle of novel ultra-fast chromatography, description of new sub-2 μm particles and related new column chemistry and technology (Mellors 2004), full-scale hardware, new designed UV detector, and connection with MS detectors (Plumb et al. 2004) was published in 2004. These columns, Acquity UPLC BEH C18 (Waters), packed with 1.7 μm particles are based on BEH (bridged ethylene hybrid) technology (Swartz et Murphy 2005). Between silanol groups are inserted hybrid ethylene bridges (see Fig. 4), which function as end-capping groups. Furthermore, the bridges fix the overall structure of this silica gelbased phase. This endows the columns with excellent mechanical robustness (max. pressure limit 15000 PSI) and chemical stability in a wide pH range. These columns can therefore be used in alkali pH range up to pH 12; acidic range up to pH 1-2, but this value depends on the column ligand used. In addition, these columns are resistant to “column bleeding”. A wide range of columns with various ligands is currently available on the market. They include BEH C18 and C8 (linear alkyl chain for classical reverse chromatography), BEH Shield RP18 (alkyl chain with inserted polar carbamate group, which combines hydrophobic and hydrophilic properties of the column and is convenient for polyphenolic compounds), BEH Phenyl (phenyl ligand on C6 alkyl chain convenient for the compounds with benzene nucleus), BEH Amide column (amidic group that retains well compounds too polar for reverse chromatography), BEH HILIC (BEH particles without a ligand, convenient for extremely polar compounds; elution uses aqueous phases with low organic content), etc. The lengths of these columns are 2, 3, 5, 10, and 15 cm, the most commonly used column has 5 cm. The inner diameter of the columns is mostly 2.1 mm and 1 mm when connected with UV and MS detector, respectively. Like the classical column, the column is protected by a pre-column and additionally by a special filter, which protects the column from potential impurities from the mobile phase. The small particle size poses demands on both the mobile phase and sample preparation. Mobile phase must be filtered through 0.2 μm membrane filter and the sample has to be filtered or centrifuged prior to analysis.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_4_Znazorneni_chemicke_struktury_hybridni_BEH_castice_1ef4bff896/Obr.-4.-Znazorneni-chemicke-struktury-hybridni-BEH-castice.jpg" alt="Fig. 4 Illustration of the chemical structure of the hybrid BEH particle with hybrid ethylene bridges"/></p>
<p>An increasing number of UHPLC columns with particle size ranging from 1.5–2 μm (e.g. Waters – Acquity BEH columns, Restek – Pinnacle® DB columns, Grace – Vision HT columns, Thermo Scientific – Hypersil GOLD, Agilent – Eclipse Plus C18 a StableBond SB-C18, Perkin Elmer – Brownlee Analytical DB C18), and the rising number of UHPLC instrument producers give evidence that UHPLC was accepted by analytical public. Apart from the Waters company (Acquity), which was the pioneer of this technology, other producers of UHPLC chromatographs with similar parameters are on the market, e.g. Agilent (1200RRLC, Infinity), ThermoElectron (Acella High Speed), Shimadzu (ProminenceUFLC), Dionex (RSLC UltiMAte 3000), Perkin Elmer (Flexar). Two synonymous terms, UPLC and UHPLC, can be found in the literature. Since UPLCTM (Ultra Performance Liquid Chromatography) technology was applied as the first in the world by the Waters Company, the term UPLC can be taken as a trade mark. Hence, UHPLC was accepted as the common term for this technology.</p>
<p>The advantages of UHPLC have been made use of also in the analysis of brewing materials and beer. Many applications were developed by the research team from RIBM (Research Institute of Brewing and Malting). A recent application, which was published in an international scientific journal in 2011, is the determination of ochratoxin A in brewing raw materials and beer (Běláková et al. 2011) and also in wine. The transformation of the original HPLC method to the new UHPLC method brought a reduction of the analysis time from the original 10 min to 2 min. The same result was obtained for the method of ferulic acid determination in barley and malt, in which the original analysis time of 12 min was reduced to 2 min (Běláková et al. 2010). The same authors developed more UHPC methods, which are used in RIBM in many applications. These include, apart from the determination of ferulic acid, also methods for the determination of other phenolic acids (cumaric acid etc.), vitamin E and vitamins from the B group in beer and brewery raw materials, patulin in beer, brewery raw materials, wine and nonalcoholic beverages, and/or determination of silymarine in an extract of Silybum Marianum.</p>
<p>In the following issues of this journal we shall present a more detailed comparison of HPLC and UHPLC method illustrated on examples of the determination of α- a β- acids in hop and hop products and determination of iso-α-acids in beer developed in RIBM.</p>
<h3><strong>3 CORE-SHELL PARTICLE COLUMNS</strong></h3>
<p>Even if the user cannot purchase a new UHPLC instrument and is bound to use a current HPLC instrument, he can still speed up the separation process. He can use columns with “fused-core silica” or synonymous “core-shell particles” working at common pressures (max 6000 PSI) that are compatible with the majority of HPLC instruments. The particles (see Fig. 5) are composed of a solid core (about 1.7 μm) surrounded by a thin porous silica layer (about 0.5 μm); their exact dimensions vary depending on the producer. The analyte can diffuse only into the pores of the thin porous ± 0.1 μm layer, where mass transfer takes place. The diameter of these 2.7 μm particles results in the generation of lower back pressure than in the case of “sub-2 particles”. At the same time, the thickness of the porous silica layer is comparable with the diameter of “sub-2 particles”. These characteristics of the “core-shell particles” ensure separation efficiency and resolution similar to UHPLC. Lastly, the analyte does not have to diffuse to the rigid core through the whole particle, which results in a shortening of diffusion path, the delay in the column, and in reduction of analysis time without deterioration of separation parameters.</p>
<p>Many papers published since 2010 have confirmed both the broad utility of core-shell columns in practice and their separation properties comparable with those of “sub-2” particles (Abrahim et al. 2010, Tylová et al. 2011). These columns, having a wide spectrum of various ligands, e.g. Halo (Mac-Mod Analytical), Ascentis Express (Sigma-Aldrich), Kinetex (Phenomenex) and/or Poroshell (Agilent) are currently available.</p>
<p>Determination of quinolones in eggs is an example of application of core-shell columns in food analysis. The column Kinetex was used and analysis time was reduced by half (Jiménez et al. 2011). The same column was also used for the first time for beer analysis for determination of iso-α-acids and some reducing forms of tetrahydroiso-α-acids (Koerner et al. 2011).</p>
<h3><strong>4 MONOLITHIC COLUMNS</strong></h3>
<p>Another approach to accelerating chromatographic process is the use of monolithic columns, which are gaining increasing popularity. In contrast to conventional stationary phases, which are formed by individual sorbent particles of defined size, the monolithic columns are formed by a single piece of porous material completely filling the interior of the column. Thus, monolithic columns do not contain any space between particles through which the majority of the flow proceeds in conventional columns. That’s why the mobile phase has to pass through the monolith pores that are much bigger than the pores in columns with common particles. Monolithic columns have two types of pores. Macropores facilitate fast convective flow of mobile phase through the monolith and significantly accelerate mass transfer between mobile and stationary phase. Mesopores provide sufficiently large surface and also a high separation capacity. This structure allows monoliths to operate at considerably high flow of mobile phase without excessive pressure increase (McCalley 2002) and without losses of separation efficiency even for separated macromolecules (peptides, synthetic polymers) (Švec 2009). </p>
<p>Monolithic columns are synthetized by hydrolytic polymeration of tetramethoxysilane or tetraethoxysilane in aqueous solution of acetic acid in the presence of polyethylene glycol. Manufacturing technology enables monolith preparation with accurately defined structure and with macropores and mesopores with convenient and adjustable sizes. The monolithic stationary phase was prepared, described and applied by, e.g., Prof. Tanaka’s group (Minakuchi et al. 1996). Many of these columns are already commercially available, e.g. Chromolith (Merck) or Onyx™ (Phenomenex in the licence of Merck). </p>
<p>As follows from the real applications and comparative studies of monolithic and conventional reversed phase columns, these columns have also found application in the area of fast chromatography and the number of their users continually increases (Samanidou et al. 2004, Nováková et al. 2004). The technology starts to be used also in food analysis. Tzanavaras et Themelis (2007) published a method of simultaneous determination of caffeine, theobromine and theophylline in coffee and non-alcoholic beverages. The analysis time did not exceed 1 min and the retention time of the last eluting caffeine was 0.7 min.</p>
<h3><strong>5 CONCLUSION</strong></h3>
<p>The huge quantity of published articles gives evidence that in the last decade UHPLC has become a favorite and widespread separation technique. It follows from these article that UHPLC is used for the separation of both small and large organic compounds such as proteins and peptides. The published data confirm the presumed advantages of UHPLC such as its high speed, sensitivity and good resolution. UHPLC technique found its use also in food analysis and begins to spread into the area of brewing and malting.</p>


New Trends in Liquid Chromatography and Their Utilization in Analysis of Beer and Brewery Raw Materials. Part 1. Theoretical Introduction

<p><strong>The new UHPLC method was compared with the HPLC method, which is used for iso-α-acid determinations in RIBM. The comparison was performed with respect to the speed of analysis, separation efficiency, mobile phase consumption, and finally repeatability. The results of this study determined all the expected advantages of the UHPLC; while maintaining the resolution this method reduced analysis time 2.5 times compared to the previous HPLC. In addition, the UHPLC reduces the consumption of organic solvents 10 times. Apart from the economic benefits the UHPLC provides a high precision of the measurement, which was verified as repeatability of the UHPLC for both forms of iso-α-aids. Especially the trans-iso-α-isomers are significant indicators of beer aging during storage and transport. It was found that the repeatability of UHPLC for the trans-isomers is better than the repeatability of HPLC.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>Iso-α-acids are formed during the brewing process in two steric forms, cis- and trans-, in a ratio of 2:1 in favor of the more stable cis-form. The factors influencing the beer aging process cause a faster decrease of the trans-form, which results in both a decrease of beer bitterness and the formation of sensory undesirable compounds. Therefore modern technology processes, which look for the ways of slowing down beer aging, focus on the possibility how to reduce the drop in the concentration of iso-α-acids (De Cooman et al., 2000).</p>
<p>During the development of analytical methods for determination of cis-/trans-iso-α-acids, close attention is paid to the isolation of these compounds from beer (SPE extraction) and to the complete separation and sufficiently sensitive determination in the beer using the liquid chromatography methods. In the present, classical HPLC methods are used for the purpose. However, new chromatography techniques based on separation on columns packed with sub-2 μm particles are being increasingly used in the analysis of beer. The efficiency of these columns does not change when the linear velocity increases. The chromatography employing these columns is called Ultra High Performance Chromatography (UHPLC). The principles of this method were described in issue 2, 2012 of the journal. An identical and often better separation can be achieved using UHPLC in comparison with HPLC; furthermore, the analysis time is significantly shorter. The higher backpressure, which is caused by extremely small particle size in UHPLC columns, has been overcome using special and newly developed pumps, absolute narrowness of the whole system, and a number of technical elements forming the new UHPLC system. Apart from the separation speed a further advantage of UHPLC is very low consumption of organic solvents (up to 10-fold lower compared to HPLC), and a very small injection volume (1–2 μl).</p>
<p>The UHPLC was used for the development of a new method of cis- and trans-iso-α-acids determination in beer. The separation parameters were compared with the classical HPLC method, which is used in RIBM (Research Institute of Brewing and Malting). Besides comparing the speed of analysis and separation efficiency the values of repeatability (r₉₅) of the two methods were confronted.</p>
<p>The separation of iso-α-acids and their isomers in beer is usually performed on the reversed C18 stationary phase. The mobile phase is prepared as a buffer with acid pH or the aqueous solution of any acid. Acetonitrile (ACN) is usually used as organic modifier. The development of highly resistant stationary phases in a wide pH range reaching up to alkali values gives rise to new methods of separation using this pH range (Hofta et al., 2007). The good selectivity of these columns enables the simultaneous determination of iso-α-acids, their oxidative products, and different forms of hydrogenated iso-α-acids. The HPLC method of determination of cis- and trans- isomers of iso- α-acids under neutral or weak alkali conditions (Harms et Nitzsche, 2001) was used in the study concerning the cis-/trans- ratio changes during beer storage under various temperature conditions. (Straková et al., 2007). However, it is necessary to note that alkali pH causes the transformation of α-acids to iso-α-acids and thus a shift in the equilibrium (Basařová et al., 2010). The dependence of isomerization on the analysis at alkaline pH has not yet been documented.</p>
<p>The original variant of mobile phase with acidic pH was chosen for the purpose of our study.</p>
<h3><strong>2 MATERIAL AND METHODS</strong></h3>
<p>Domestic beers with the bitterness ranging from 25 to 34 mg/l were chosen for comparing the efficiency of UHPLC and HPLC methods in the determination of cis- and trans-iso-α-acids. The iso-α-acids were isolated from the beer using the SPE technique on the extraction columns Phenomenex according to a previously described procedure (Jurkova et al., 2003).</p>
<p>The iso-α-acid dicyclohexylamine containing 62.3% of iso-α-acids and highlighted as DCHA-Iso, ICS-13, which is an international calibration standard (ICS), was used as an external standard for the calibration in both methods.</p>
<p>The stock solution of the calibration standard was prepared by dilution of ICS-13 (20 mg) weighed with the precision of 0.1 mg in 100 ml of gradient grade methanol (99.8%, Merck). Prior to the dilution, the methanol was acidified with phosphoric acid (p.a. Merck) in an acid-methanol ratio of 0.5 : 1000 (v/v). For HPLC and UHPLC, the calibration standard was diluted tenfold and twentyfold with acidified methanol, respectively.</p>
<h5><strong>HPLC chromatographic conditions</strong></h5>
<p>The separation was performed on a reversed phase column (Alti-ma C18, 5 μm, 150 x 4.6 mm, Alltech) protected by a C18 pre-column (4 x 3 mm, Phenomenex). The linear gradient was applied and two-component mobile phase (A) and (B) was used. Phase (A) with pH 2.7 was prepared from ultrapure water (Millipore) with a maximum organic compound content of 5 ppb, and phosphoric acid. Phase (B) was gradient grade acetonitrile (≥99.9%, Sigma Aldrich). The linear gradient started with 52 % of ACN, the content of ACN linearly in-creasing within 30 min to the final concentration of 67 %. The equilibration time was 10 minutes. The flow rate was 1.5 ml/min and the column temperature was 40 °C. The analytes were detected in the UV area at 275 nm. The injection volume was 10 μl.</p>
<p>The Spectra SYSTEM (TSP, USA) liquid chromatograph with PDA detector was used for the analysis. The data collection and data processing was carried out with the ChromQuest software for Windows NT.</p>
<h5><strong>UHPLC chromatographic conditions</strong></h5>
<ul>
<li><p>UHPLC analyses were performed on the BEH C18 column (2.1 x 50 mm I.D., 1.7 μm), Waters, the column temperature was 40 °C and the mobile phase flow was 0.4 ml/min. The diode array detector (PDA) was used for detection; analytes were detected at 275 nm. The two-component mobile phase was composed of acetonitrile (B) and aqueous solution of acids or buffers (ultrapure wa-ter, Millipore) with pH ranging from 1.6 to 3.08 (A). During the optimization the following mobile phase (A) were tested:</p>
</li>
<li><p>TFA, pH 1.6 (0.1% vol., prepared as a solution of 100 μl TFA, 99.95% ULC/MS, Biosolve, Netherlands) in 100 ml of ultrapure water;</p>
</li>
<li><p>TFA, pH 2.7 (0.01% TFA vol., prepared as a solution of 10 μl, 99.95% ULC/MS, Biosolve, Netherlands) in 100 ml of ultrapure water; using of this conditions was achieved the best separation, thus,</p>
</li>
</ul>
<p>this phase was finally chosen as optimal (see section “3. Results and Discussion”);</p>
<p>Ammonium formate, 5 mM, pH 3.08 (prepared using titration of 5 mM formic acid (99%, Merck, Germany) with aqueous solution of ammonium hydroxide, 29%, A.C.S. reagent, Sigma Aldrich, Germany to the final pH of 3.08).</p>
<p>The gradient elution was optimized in terms of the minimal analysis time and maximal resolution of determined compounds. The final gradient conditions were as follows: the gradient started at 48% of phase (B) and grew linearly up to 55% (B) within 11 min. The subsequent isocratic step at 55% (B) and equilibration step starting at final condition of 48% (B) were 2 min and 3 min, respectively. The injection volume was 2 μl.</p>
<p>UHPLC analyses were performed on Acquity UPLCTM (Waters) chromatograph with the 2996 PDA detector operating in the range from 194 to 600 nm. Data were processed with Empower 2 software (Waters). Chromatograms were extracted at 275 nm before interpretation, data sample rate was set at 20 pts/s and filter constant was 0.5.</p>
<h3><strong>3 RESULTS AND DISCUSION</strong></h3>
<p>Comparison of UHPLC and HPLC chromatograms of separated iso-α-acids isomers is given in Fig. 1 and Fig. 2, respectively. Both chromatograms were measured under optimal UHPLC and HPLC conditions described in Materials and Methods. Fig. 1 confirms that the UHPLC method is faster than HPLC; the analysis time was reduced 2.5 times in comparison with HPLC method. In the UHPLC, the last eluting peak of cis-iso-ad-humulone had the retention time of 4.9 min, the total analysis time including the wash step was 13 min, and equilibration step was 3 min. Thus, the total analysis time was 16 min. In the HPLC, the last eluting peak of cis-iso-ad-humulone eluted at 12 min, the gradient wash step lasted 30 min, and the equilibration step was 10 min, the total HPLC analysis time being thus 40 min. In both events, the gradient is continuing also after elution of cis-iso-ad-humulone because residual α- and β-acids can elute even later. If the detection of α- and β-acids is not required (reduced method version) then it is possible to reduce the gradient by implementing a fast wash step with a high content of organic modifier after 5th and 12th min in UHPLC and HPLC mode, respectively (see Fig. 1 and 2). If the wash step is not included or if it is not sufficient, the residual α- and β-acids can accumulate on the column and can subsequently interfere in the following analyses. The comparison revealed a noticeable difference between the two methods. Apart from the marked difference in the time of analysis a huge difference in the consumption of organic solvents is evident. While the ACN consumption under our experimental conditions in the UHPLC mode is 32 ml per 10 samples, in the HPLC mode it is 360 ml per 10 samples, i.e. ten times more.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Kvasny_prumysl_Obr_1_UHPLC_chromatogram_realneho_vzorku_piva_75c1ba51b2/Kvasny-prumysl-Obr.-1-UHPLC-chromatogram-realneho-vzorku-piva.jpg" alt="Fig. 1 UHPLC chromatogram of real beer sample"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Kvasny_prumysl_Obr_2_HPLC_chromatogram_realneho_vzorku_piva_775417f163/Kvasny-prumysl-Obr.-2-HPLC-chromatogram-realneho-vzorku-piva.jpg" alt="Fig. 2 HPLC chromatogram of real beer sample"/></p>
<p>The transformation of the HPLC method to the UHPLC and its optimization revealed a strong dependence of the retention time of studied compounds, iso-α-acids, on the mobile phase pH. Even small changes in the monitored pH range (from 1.6 to 3.08) give rise to a significant shift of the retention under the maintenance of the organic modifier gradient. A very good separation was achieved in UHPLC mode at pH 2.7, when the retention time of the last eluting peak belonging to iso-α-acid (cis-iso-ad-humulone) was 4.9 min. This pH proved as optimal even for usage in HPLC mode, where the retention time of this peak is 12 min. When the pH was decreased to 1.6 (0.1% TFA) the retention times was extended four times and the last determined peak of cis-iso-adhumulone eluted at 19th min. Additionally, the peaks of trans-iso-ad-humulone and cis-iso-n-humulone were not separated. On the contrary, when the pH was increased to 3.08 (5 mM ammonium formate) the retention times were notice-ably reduced. All iso-α-isomers eluted in the range from 1 to 2 min, but isomeric forms cis- and trans- of any acids were not separated.</p>
<p>TFA, formic acid, and phosphoric acid were tested as potential additives to the aqueous part of the mobile phase. Phosphoric acid is commonly used in the original HPLC method. However, this acid was found to be unsuitable in the UHPLC mode, because all the peaks were considerably deformed. Finally, TFA proved to be the optimal additive because its usage showed the best separation parameters. During the gradient optimization it was necessary to decrease the initial content of organic modifier to 48%, because the use of the same percentage of ACN as in HPLC (52 %) caused fast peak elution with insufficient separation. This observation is in correlation with the UHPLC theory according to which the analytes pass through the column under much higher pressure (up to five times) compared to HPLC. The final content of ACN in the gradient was also changed, the percentage of 55 % being sufficient for good and fast separation instead of the original 67 %. Finally, the time of duration of the linear gradient was also reduced, specifically from 30 to 11 min.</p>
<p>The last step was the optimization of the injection volume. Regard-ing the different column dimension of 150 x 4.6 mm and 50 x 2.1 mm in HPLC and UHPLC, respectively, the injection volume should be reduced during the method transfer. Injection volume higher than 2 μl caused overloading of UHPLC column, which appeared in the peak splitting. When 2 μl of the sample was loaded on the column it ensured a very good separation of all determined compounds with convenient sensitivity which was in accordance with the quantification limits of the method.</p>
<p>The repeatability of the new UHPLC method was compared with the original HPLC method and was calculated from the values of the iso-α-concentration values in 4 real beer samples, every sample was measured in duplicate. The evaluation was carried out from the peak areas of both trans- and cis- forms of iso-co-humulone, iso-ad-humulone, and iso-n-humulone. The results of individual concentrations and repeatability values of all studied isomers in both UHPLC and HPLC systems are given in Tab. 1 and 2. The repeatability r₉₅ was calculated according to the formula r₉₅ = sᵣ x 2,8, where sr is standard deviation calculated for various, but similar, samples measured un-der the conditions of repeatability:</p>
<p>sᵣ² = ΣDᵢ²/2n, where Dᵢ is the range of values from two determinations.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Kvasny_Prumysl_Tab_1_Stanoveni_cis_a_trans_forem_iso_a_kyselin_metodou_HPLC_80e5e9cb6f/Kvasny-Prumysl-Tab.-1-Stanoveni-cis-a-trans-forem-iso-a-kyselin-metodou-HPLC.jpg" alt="Tab. 1 Determination of cis- and trans- forms of iso-α-acids using the HPLC method"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Kvasny_Prumysl_Tab_2_Stanoveni_cis_a_trans_forem_iso_a_kyselin_metodou_UHPLC_cff3f07790/Kvasny-Prumysl-Tab.-2-Stanoveni-cis-a-trans-forem-iso-a-kyselin-metodou-UHPLC.jpg" alt="Tab. 2 Determination of cis- and trans- forms of iso-α-acids using the UHPLC method"/></p>
<p>The UHPLC method affords a comparable or even better repeat-ability compared to the HPLC for the all cis- and trans-iso-α-acids with the exception of cis-isomers of co-humulone and n-humulone. This could be probably caused by better geometry of UHPLC peaks leading to better peak integration and thus, better quantification.</p>
<p>Furthermore, it could be established that the use of the UHPLC method results in a smaller variance and thus a more accurate con-centration determination of trans- forms, which are considered as an indicator of beer stability or beer ageing during storage or transport.</p>
<p>The Tab. 3 summarizes described differences and advantages of newly developed UHPLC method comparing the original HPLC method.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Kvasny_Prumysl_Tab_3_Srovnani_parametru_metod_UHPLC_a_HPLC_45ebc16b36/Kvasny-Prumysl-Tab.-3-Srovnani-parametru-metod-UHPLC-a-HPLC.jpg" alt="Tab. 3 Comparison of UPLC and HPLC method parameters"/></p>
<h3><strong>4 CONCLUSION</strong></h3>
<p>The new generation of UHPLC liquid chromatography based on more advanced instrumental technique is not only more economical (reduced analysis time with low consumption of solvents) but it also shifts the analysis of beer to the area with more accurate measurement and lower variance of results, which was demonstrated in this study on the example of determination of steric iso-α-acid isomers.</p>


New trends in liquid chromatography and their utilization in analysis of beer and brewery raw materials.  Part 2. Determination of cis/trans- isomers -iso-α-acids in beer using Ultra Performance Liquid Chromatography

<p><strong>The traditional method of High Performance Liquid Chromatography (HPLC) was compared with the currently increasingly used Ultra-High Performance Liquid Chromatography (UHPLC) on a representative group of 11 hop samples. The results of this work confirmed that the UHPLC technique ensures high throughput also in determination of α- and β-acids. Another benefit of this method is the decrease of result uncertainty which is determined by high separation properties of the UHPLC system. While original HPLC method originating from Analytica EBC 7.7 provides 4 separate peaks (co-elution of n-adhumulone and adhumulone and also of n-lupulone and adlupulone), all 6 analyzed bitter compounds were completely separated using UHPLC.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>The first step of all types of analyses is the extraction of α- and β-acids (soft resins) into a nonpolar solvent. At the beginning, the titration method used the extraction into toluene (Analytica-EBC, 2005a), but this solvent was later replaced by diethylether in acidic conditions (Analytica-EBC, 2005b). The extract is titrated with lead acetate leading to formation of complexes with α-acids and the conductivity of this system is measured at the same time. The content of α-acids in the hop is expressed as % (w/w) alias LCV (lead conductance value). This value summarizes the content of α-acids, however, the reaction is less specific and the determined value is less accurate in the presence of any compounds reacting with lead acetate. The proportion of β-acids is approximately the difference between the total content of soft resins and the LCV value. Ethereal extract can be analyzed by HPLC following its centrifugation and dilution in methanol. Chromatographic method enables either complete or partial separation of α-acid zones, cohumulone, n-humulone, and adhumulone. The efficiency of the separation is dependent on the chromatographic column and mobile phase used. A similar chromatographic behavior was recorded in case of β-acids, colupulone, n-lupulone, and adlupulone. In contrast to titration methods, the HPLC methods are more specific and provide results better corresponding to real soft resins quantity. </p>
<p>It is evident that accuracy and precision of determination of α-acids in hop and hop extract affect the marketing and trade relations. Hence, all subjects as breweries, growers, and traders, are interested in accurate and comparable results of analytical laboratories. Good reproducibility and accuracy of results obtained by HPLC methods is ensured by using unified international calibration standard ICE3, which is presently already the third in a series. European laboratories follow the methods of European Beer Convention (Analytica EBC, 1998) and they can regularly participate in international proficiency testing organized by MEBAK. RIBM takes part in proficiency testing organized by MEBAK regularly two times a year and in BAPS (Brewing Analytes Proficiency Testing Scheme) regularly six times a year. Additionally, methods for hop analysis are accredited by the Czech Accreditation Institute (CIA).</p>
<p>EBC methods for α- and β-acids are developed for the most commonly used HPLC systems. In parallel with the development of liquid chromatography instrumentation the UHPLC systems start replacing it in the analytical field. The benefit of this new system is not only reduction of analysis time and consumption of organic solvents, but also more repeatable results with considerably lower uncertainty (Olšovská et al., 2012b). Information about UHPLC was described in more detail in previous issues of this journal (Olšovská et al., 2012a,2012b). The total analysis time of the content of bitter acids in the hop could be eventually reduced by an effective pressure extraction procedure, which was developed also at RIBM (Čulík et al., 2009). The rapid extraction together with modern UHPLC technique could bring to laboratories the possibility of sample high-throughput, and, moreover it could ensure more accurate data on the content of α-acids for traders in the hop commodity.</p>
<h3><strong>2 MATERIAL AND METHODS</strong></h3>
<p>Hop samples with the content of α-acids ranging from 1.4 to 8.5 % (w/w) were used for comparison of UHPLC and HPLC methods. The samples were extracted and prepared for LC analysis according to EBC method 7.7 (Analytica – EBC, June 2005).</p>
<p>The international calibration standard ICE3 was used for the method calibration; the total content of α- and β-acids was 44.64 % (w/w) and 22.28 % (w/w), respectively. The calibration solution was prepared as follows. ICE3 (0.5 g) was weighed with 0.1 mg precision, dissolved in 100 ml methanol, and diluted ten times with methanol (Gradient Grade, Sigma Aldrich).</p>
<h5><strong>Chromatographic conditions HPLC</strong></h5>
<p>α- and β-acids were analyzed on chromatographic column with C-18 Hop reversed phase (250 x 4 mm, particle size 5 μm), Machery Nagel, Germany. The separation was in isocratic mode. The mobile phase was a mixture of 850 ml of methanol (for HPLC, Sigma Aldrich), 190 ml of water (Millipore, purity max. 5 ppb of organic compounds) and 5 ml of phosphoric acid (analytical grade, Merck). The column temperature was 35 °C, the flow rate was 0.8 ml/min, and the injection volume was 5 μl. α- and β-acids were detected in UV at 314 nm according to Analytica EBC.</p>
<p>Analyses were performed on SpectraSYSTEM liquid chromatograph (TSP, USA) with diode array detector (DAD). The data collec tion was carried out using chromatography software ChromQuest for Windows NT.</p>
<h5><strong>Chromatographic conditions UHPLC</strong></h5>
<p>UHPLC analyses were performed on BEH C18 Waters column (2.1 x 50 mm I.D, particle size 1.7 μm), column temperature was 25 °C, and mobile phase flow was 0.4 ml/min. The two-component mobile phase was formed by aqueous solution of TFA-water (0.1:99.9 v/v) (A) and acetonitrile (B). The separation of analytes was carried out by gradient elution from a starting point at 50 % (B) to 80 % (B) within 6 min. The analytical run was terminated with an equilibration step of 2 min. The injection volume was 2 μl. α- and β-acids were detected by UV at 314 nm. </p>
<p>The Acquity UPLCTM (Waters) chromatograph was used for the analyses together with 2996 PDA detector operating at wavelengths ranging from 194 to 600 nm. Data were processed by Empower 2 (Waters) software.</p>
<h3><strong>3 RESULTS AND DISCUSSION</strong></h3>
<p>HPLC chromatogram obtained according to EBC 7.7 method is depicted in Fig. 1. α-Acids elute in two zones; cohumulone elutes in a discrete zone whilst n-humulone and adhumulone co-elute in a common zone. A very similar elution profile was obtained when β-acids were separated. When the UHPLC method was optimized, a complete separation of all three zones was obtained for α- and β-acids (see Fig. 2). An absolutely different type of column was used in UHPLC mode compared to HPLC. This column is based on the principle of “sub-2 μm” and we described this special column type in our previous article (Olšovská et al., 2012a). In the literature one can usually encounter the phenomenon of multiple increase of performance of UHPLC columns as compared with HPLC columns. Using of a new UHPLC method with modified mobile phase (with a higher content of more polar component) lead to a total separation of peaks in the case of n-humulone and adhumulone (and analogously n-lupulone and adlupulone), which were not at all separated in HPLC. Furthermore, analysis time significantly decreased from 25 min in HPLC to 8 min; this facilitates high-throughput of samples and cost reduction of solvent consumption. A very simple mobile phase, 0.1 % TFA in water (v/v) and acetonitrile, was used in the UHPLC mode. The consumption of organic modifier was minimal due to the speed of analysis (approximately 2 ml per one analysis including equilibration step), which leads to the price reduction of the method. When bitter acids were analyzed by HPLC, 17 ml of organic modifier (methanol) was used.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_1_HPLC_chromatogram_extraktu_chmele_3b863645de/Obr.-1-HPLC-chromatogram-extraktu-chmele.jpg" alt="Fig. 1 HPLC chromatogram of hop extract (Sladek variety) Chromatographic conditions: Column C18-Hop (250 x 4 mm, particle size 5 μm), Macherey Nagel; column temperature 35 °C; mobile phase flow 0,8 ml/min; mobile phase composition 850 ml of methanol, 190 ml of water and 5 ml of phosphoric acid; isocratic elution; injection volume 5 μl; UV detection at 314 nm"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_2_UHPLC_chromatogram_extraktu_chmele_ed5f6da57b/Obr.-2-UHPLC-chromatogram-extraktu-chmele.jpg" alt="Fig. 2 UHPLC chromatogram of hop extract (Sladek variety) Chromatographic conditions: Column BEH C18 (2.1 x 50 mm I.D, particle size 1.7 μm), Waters; column temperature 25 °C; mobile phase flow 0,4 ml/min; mobile phase composition (A) TFA–water (0.1:99.9, v/v), (B) acetonitrile; gradient elution from 50% (B) to 80% (B) within 6 min, equilibration 2 min; injection volume 2 μl; UV detection at 314 nm"/></p>
<p>The UV spectrum of α-acids shows three maxima at 237, 285, and 322 nm (see Fig. 3). The wavelength of 314 nm and one-point calibration was used according to EBC. 7.7. The content of α- and β-acids was measured by HPLC and UHPLC under these convention conditions in 11 hop samples. Tab. 1 gives an overview of analyzed hop samples together with LCV values. The values of total α- and β-acids content obtained by the two chromatographic methods together with their difference Δ are summarized in Tab. 2. The theoretical value of repeatability r95 calculated for individual levels follows from EBC 7.7 and expresses the permitted difference between two parallel results in one laboratory. The values of real and theoretical difference derived from method repeatability were compared. As shown in Tab. 2, UHPLC gives moderately higher results in case of α-acids (on average 0.13 % higher). Nevertheless, for all samples (except for one case) these differences are lower than the permitted repeatability. Both positive and negative differences were found with α-acids; except for one case they were again below theoretical repeatability.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_3_UV_spektrum_kohumulonu_664083c214/Obr.-3-UV-spektrum-kohumulonu.jpg" alt="Fig. 3 UV spectrum of cohumulone The UV spectrum was measured using UHPLC-DAD detector during the separation of α-acids under conditions mentioned in Materials and Methods. The other α-acids had the same UV maxima at 237, 285 a 322 nm"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_1_Prehled_testovanych_vzorku_chmele_d9bc551232/Tab.-1-Prehled-testovanych-vzorku-chmele.jpg" alt="Tab. 1 An overview of tested hop samples and their lead conductance value (LCV)"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_2_Srovnani_obsahu_a_a_v_kyselin_c2a48394b3/Tab.-2-Srovnani-obsahu-a-a-b-kyselin.jpg" alt="Tab. 2 Comparison of the content of α- and β-acids in tested hop samples obtained by HPLC and UHPLC"/></p>
<p>The two methods can thus be considered comparable. Two hop samples (Sladek and Saaz) with different concentration levels of α- and β-acids were chosen for comparison of validation parameters. Both hop samples were measured repetitively (n=8) chromaand the repeatability and relative uncertainty estimate (k=2) were calculated. The Sladek variety (KH 10.18) contained 8.57 ± 0.1 % (w/w) and 3.81 ± 0.04 % (w/w) of α- and β-acids, respectively. The Saaz variety contained 2.81 ± 0.03 %(w/w) and 5.57 ± 0.06 % (w/w) of α- and β-acids, respectively. As follows from Tab. 3, the repeatability and relative uncertainty estimate for α- and β-acids are significantly (more than ten times) lower in all cases when the UHPLC is used.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_3_Srovnani_validacnich_charakteristik_HPLC_a_UHPLC_metody_b61c4f6b0e/Tab.-3-Srovnani-validacnich-charakteristik-HPLC-a-UHPLC-metody.jpg" alt="Tab. 3 The comparison of HPLC and UHPLC validation parameters"/></p>
<h3><strong>4 CONCLUSIONS</strong></h3>
<p>This study is the last of a series of three studies, which dealt with a new generation of chromatographic columns and related liquid chromatography instrumentation known as Ultra High Performance Liquid Chromatography (UHPLC). In the first part, we introduced this new technology and described its enormous advantages over the classical HPLC method, which result from the basic principle of UHPLC instrumentation, and which will undoubtedly positively reflect in economical costs per analysis. Two useful studies were performed in the area of beer analysis to verify this claim.</p>
<p>The usefulness of the new method was confirmed on the example of iso-α-acid determination in beer and further on determination of α- and β-acids in hop. Besides an economic benefit (reduction of time, energy and organic solvents as a result of significant analysis time reduction) this method affords measurement with lower variance of results. The current methods of beer analysis are based on some conventions that presume the use of old HPLC instrument types. Based on both studies it is possible to state that the use of UHPLC provides comparable results with HPLC, when defined measure parameters (the way of detection, calibration and quantification) are maintained. Therefore, both types of LC can be used without compromising the accuracy of the results.</p>


New Trends in Liquid Chromatography and Their Utilization in Analysis of Beer and Brewery Raw Materials. Part 3. Comparison of HPLC and UHPLC Determination of α- and β -Acids

<p><strong>The compounds with the same electrochemical manners as epicatechin and catechin and oligomeric proanthocyanogens (OPCs) related to catechin were distinguished from the other compounds according to their electrochemical responses on lower potential (500 mV). The total content of these OPCs including catechin and epicatechin was determined as an equivalent of concentration of the catechin used as an external standard. The results were correlated partly with the results obtained by using of the group analysis for determination of the total polyphenol and for the determination of the anthocyanogens, partly with results obtained by using of the modern method electron spin resonance (ESR) for determination of the antioxidant activity.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>The hop plant (Humulus lupulus L.) from Cannabinaceae family contains beside primary compounds responsible for basic sensory features (bitterness, fragrance and fullness) also many compounds of health importance. There are especially a group of dimers and trimers, antioxidants (1) derived from catechin and epicatechin, socalled oligomeric proanthocyanogens (OPCs) and a group of prenylated flavonoids in hops (2.3), of which xanthohumol is the most abundant 0.5–1.1 %).</p>
<p>The both groups of these compounds have healing ability and protect human organism against development of many civilization diseases. The group of antioxidants OPCs with lower potentials to their oxidation shields the organism against oxidation of DNA and so protect human organism against tumor diseases (4). Flavonoids are perceived by human receptors like hormon oestrogen, thus these compounds show the oestrogenic effect (5,6). The oestrogenic activity of these compounds is using to the menopause anesis and thus return the organism to normal state. These compounds are also very effective for osteoporosis therapy. Prenylflavonoids are oxidized by higher potential (daidzein, genistein) so their contribution to antioxidant processes is lower and their main activities are interactions with tissue receptors influencing the oestrogenic activities and regulation of cell division (7).</p>
<p>The electrochemical detection of CoulArray detector enables a resolution of the electrochemically different groups of compounds based on their voltametric behavior.</p>
<p>The selectivity of this detection enables to analyze the target group of compounds (8) and we can distinguish analytes with lower oxidative potential 400–500 mV ( catechin and from it derived OPCs) from analytes with higher oxidative potential, e.g. ferulic acid (700 mV) or 4-hydroxybenzoic acid or 4-hydroxyphenylacetic acid (900 mV) (9).</p>
<p>The aim of this study was the determination of antioxidant activity of hop oligomeric proanthocyanogens by the method of liquid chromatography with CoulArray detection and comparison with results obtained partly by modern method electron spin reverberation (ERS), partly by classical methods for determination of total polyphenols and determination of anthocyanogens.</p>
<h3><strong>2 MATERIAL AND METHODS</strong></h3>
<p>The objects of this study were hop varieties Harmonie, Rubín and new varieties code-named 4816, 4849 and 5008. </p>
<p>With regard to sufficient polarity of oligomeric proanthocyanogens and good water solubility, we used the cold hop leaches in this study.</p>
<p>Due to complexity and unknown chemical structures of particular components of hop oligomeric proanthocyanogens so far, the fingerprint technique was chosen for evaluation. All OPCs components eluated in range from 20 to 40 min. were evaluated on potential 500 mV (Fig. 1). The sum of concentrations of all OPCs was expressed using of the concentration of the catechin used for the calibration. The values obtained in this way were correlated with values obtained by other methods: modern ESR-DPPH and classical methods for determination of total polyphenols and determination of anthocyanogens. </p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_1_Chromatogram_merene_frakce_OPC_9427a14b7e/Obr.-1-Chromatogram-merene-frakce-OPC.jpg" alt="Fig. 1 Chromatogram of measured fraction of OPCs"/></p>
<p>The samples for all analytical methods were prepared by cold leaching: 5 g of milled hops were agitated in 250 ml of distilled water at laboratory temperature for 30 minutes. The leach was centrifuged (15 minutes, 10 000 rpm) and stored by freezing before analysis.</p>
<h3><strong>HPLC</strong></h3>
<h5><strong>Sample preparation</strong></h5>
<p>Hop leach was diluted in ratio of 1:1 with mobile phase A used for chromatography separation and filtrated through cellulose syringe filter 0.20 μm (Chromafil RC-20/25, Macherey Nagel) into chromatographic vials.</p>
<h5><strong>Chromatographic conditions</strong></h5>
<p>The buffer 0.005 M ammonium acetate in ultraclean water, TOC &lt; 5ppb was used for preparation of both phases A and B. The phase A contained 5 % acetonitrile (gradient grade, Sigma Aldrich), the phase B contained 50 % acetonitrile of the same quality. Both phases were filtered through membrane filter Nylon 66, 0,2 μm (Supelco) and pH was adjusted to 3.00 by formic acid (Fluka). All chemicals were for MS application.</p>
<h5><strong>Column</strong></h5>
<p>Purospher STAR RP-18e (5 μm) 250 x 4.6 mm (Merck) Gradient: 0–10 min. 0% B, 10–18 min. 0–8 % B, 18–40 min. 8–10 % B, 40–77 min. 10–21 % B, 77–120 min 21–85 % B. The content of mobile phase B was increased to 100 %. The measuring cells were cleaned and the 15 min equlibration by phase A was applied afterwards. </p>
<ul>
<li>Temperature of column: 35 °C.</li>
<li>Flow: 0.8 ml/min.</li>
<li>Injection of the sample: 10 μl.</li>
<li>The measuring potentials setting in turn 250, 300, 400, 500, 600, 700, 800 and 900 mV.</li>
</ul>
<h5><strong>Calibration</strong></h5>
<p>The catechin was chosen as the equivalent to express the concentration of all compounds with the same antioxidant activity on reaction potential 500 mV The standard solutions of catechin 0.01; 0.1; 0.5; 1.0 and 5.0 mg/l were used for the external calibration. The determination of all OPCs was realized by the measuring and checking-out of heights of all peaks with the same electrochemical features like catechin. The concentration of OPCs was expressed as catechin (mg/l) according to external calibration. </p>
<h5><strong>Determination of the antioxidant activity DPPH</strong></h5>
<p>The antioxidant activity of individual hops was determined using the technique of electron spin resonance (ESR). The method is based on the reaction of the stable radical of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) with antioxidants present in the sample.</p>
<p>The antioxidant activity of samples (ARA 2) is expressed as the decline of the concentration of DPPH after 10 minutes of the reaction.</p>
<p>All the measurements of the free radicals were performed on the Spectrometer MiniScope MS 200, Magnettech GmbH, Germany.</p>
<p>The assay: the aliquot of 14 ml of the DPPH stock solution is mixed with 1 ml of the sample in the test tube without delay. The tube is then inserted into the autosampler which is tempered to 30 °C. The measuring program controlling the transport of the reaction mixture in the interval of 1 minute is then immediately triggered. The value of the free radical DPPH signal is then monitored during the following 10 minute interval. After completion of the analysis the measured values are processed by the particular analytical software of the spectrometer. The result of these calculations is a dependence of the value of the DPPH signal of the sample on time. The antioxidant activity ARA2 is expressed in the percent proportion of the DPPH value decline after the 10 minute reaction.</p>
<h5><strong>Determination of the total polyphenol</strong></h5>
<p>Total polyphenols were determined according to EBCAnalytica (10).</p>
<h5><strong>Determination of the anthocyanogens</strong></h5>
<p>Anthocyanogens were determined according to „Pivovarskosladařská analytika“ (11]).</p>
<h3><strong>3 RESULTS AND DISCUSSION</strong></h3>
<p>The correlations of results obtained by described electrochemical method with CoulArray detection and results obtained using methods: ESR – DPPH (R 2 = 0,9412), determination of total polyphenols (R2 = 0,9867) and determination of anthocyanogens (R2 = 0,991) were found. These correlations are shown in Fig. 2, 3, 4. Coefficients of determination were calculated using of statistical programme Microsoft Office Excel 2003.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_2_Elektrochemicka_metoda_s_Coul_Array_detekci_v_porovnani_s_metodou_ESR_DPPH_pro_kvantitativni_vyjadreni_antioxidacni_aktivity_matrice_e7cf9ce546/Obr.-2-Elektrochemicka-metoda-s-CoulArray-detekci-v-porovnani-s-metodou-ESR-DPPH-pro-kvantitativni-vyjadreni-antioxidacni-aktivity-matrice.jpg" alt="Fig. 2 Comparison of electrochemical method with CoulArray detection and ESR-DPPH method for quantification of antioxidative
activity of hop matrix"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_3_Elektrochemicka_metoda_metoda_s_Coul_Array_detekci_v_porovnani_s_metodou_pro_stanoveni_celkovych_polyfenolu_jako_mira_antioxidacniho_1b3ffab13c/Obr.-3-Elektrochemicka-metoda-metoda-sCoulArray-detekci-v-porovnani-s-metodou-pro-stanoveni-celkovych-polyfenolu-jako-mira-antioxidacniho.jpg" alt="Fig. 3 Comparison of electrochemical method with CoulArray detection and method for determination of total
polyphenols as a rate of antioxidative potential hop matrix"/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_4_Elektrochemicka_metoda_metoda_s_Coul_Array_detekci_v_porovnani_s_metodou_pro_stanoveni_anthokyanogenu_jako_mira_antioxidacniho_bc81bd706a/Obr.-4-Elektrochemicka-metoda-metoda-s-CoulArray-detekci-v-porovnani-s-metodou-pro-stanoveni-anthokyanogenu-jako-mira-antioxidacniho.jpg" alt="Fig. 4 Comparison of electrochemical method with CoulArray detection and method for determination anthocyanogens
as a rate of antioxidative potential hop matrix"/></p>
<p>The newly developed chromatographic method with electrochemical detection can be used to comparison of hops and similar materials with antioxidant activity. This method can be used for evaluation of antioxidant activity like the modern ESR-DPPH method and the classical methods for determination of total polyphenols and for determination of anthocyanogens.</p>
<p>But the best match (R2 = 0,991) was achieved with the method for determination of anthocyanogens, this group of compounds is similar to catechins and their oligomers due to structures and substituents on benzene ring. The correlative coefficients with other methods reflect the enforcement other compounds with antioxidant activity, nevertheless their values 0.9867 and 0.9412 confirm a good agreement. It can be concluded that the main groups responsible for the hop antioxidant features are catechins, their oligomers and anthocyanogens. Other structures like ferulic acid, p-hydroxybenzoic and 4-hydroxy - phenylacetic acids have the lower contribution to the antioxidant activity due to their higher oxidative potentials.</p>


Hops - an abundant source of antioxidants. Methods to assessment of antioxidant activity of hop matrix

N-nitrosamines in 21th Century

<p><strong>The plant Stevia rebaudiana (Stevia sweet) is becoming a favourite sweetener in the food industry. Sweet taste is caused by diterpenic glycosides, so-called steviol glycosides, which lack calories while being 300 times sweeter than sucrose. While all the other glycosides are slightly or even disagreeably bitter, rebaudioside A possesses desirable sensorial properties. Because this sweetener is used to sweeten beverages including mixed beer, a fast routine analytical method was developed using HPLC with UV detection for determination of rebaudioside A in mixed beer and lemonade. The HILIC principle, which is suitable for separation of polar and hydrophilic analytes, was used for separation of steviol glycosides. The method is selective, reproducible and has a high recovery of 90-100 %.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>Sweet stevia (Stevia rebaudiana) is native to South America, plentifully spread in Asia, especially in Japan and is used as a non-calorifi c sugar substitute.</p>
<p>French chemists isolated basic glycosides stevioside and rebaudioside A from plant leafs in 1931. They are diterpenic compounds with high sweetness, approximately 300 times sweeter than sucrose, and are contained in all parts of this plant with the exception of roots. Dry leaves contain about 10% of stevioside and 2–4% rebaudioside A. The stevia leaves contain altogether eight diterpenic steviol glycosides: stevioside, steviolbioside, rebaudioside (A, B, C, D, E) and dulcoside A; their concentration is minor. Diterpenic steviol glycosides have a slightly bitter taste except for rebaudioside A, which has minimal bitterness. The major produced compound, stevioside, exhibits appreciable unpleasant bitterness (Prakash et al., 2008) and rebaudioside A extract of is therefore the only one used in food and beverage industry. Rebaudioside A extracts can differ in the content and purity of the final product depending on plant origin and extraction technique. Apart from steviol glycosides, extracts of stevia can contain also other compounds without any sweetness.</p>
<p>The worldwide “stevia rediscovery” as a food supplement required the overcoming of numerous obstacles presented by lobbyist groups since it represented a significant competition to other artificial sweeteners then in use. The US Food and Drug Administration FDA recognized stevia as a food supplement only in 1995; before that sloustevia could be used only in cosmetic industry although its safe use was known more than 1500 years. The position of the FDA was influenced by the misleading studies by the Brazilian Prof. Mauro Alvarez in 1988, which described stevia as having contraceptive properties and even causing infertility of men and women. A number of studies had to be performed to disprove this myth. Nearly 40 years thus elapsed before FDA approved stevia as a common sweetener without any contraceptive effect. None of the current studies has confirmed its effect on human fertility (Kumar et al., 2008).</p>
<p>Also in European Union extensive discussions and often mutually contradictory studies preceded the recognition of stevia as food supplement with designation E960.</p>
<p>Steviol glycosides were registered by EU as permitted sweetener only on December 2, 2011. This approval concerns rebaudioside A with the content of at least 90% of pure substance and mixtures of sterol glycosides with a total content higher than 95%. </p>
<p>According to the Joint Committee of Experts for Food Additives (JEFCA) of World Health Organization (WHO) and of Food and Agriculture Organization of the United Nations (FAO) the acceptable daily intake of steviol glycosides is 4 mg of steviol per kg body mass a day (US Food and Drug Administration, 2008, JEFCA 2008). The sweetener rebaudioside A has found application in all branches of food industry as a non-calorific sweetener; it is used especially for sweetening of lemonades and syrups in beverage industry. Fruit beers sweetened with rebaudioside A have recently appeared increasingly often on the market. The slightly bitter taste of rebaudioside A harmonizes with the bitter beer character and it replaces more and more artifi cial sweeteners such as aspartam.</p>
<p>The widespread method for determination of steviol glycosides is HPLC, though there exist also some methods using thin layer chromatography, capillary electrophoresis or spectroscopic methods (NIR, VIS). The choice of HPLC separation conditions is dependent on the polar character of the glycosides, the choice of UV detection restricts the range of applicable absorbance to short wavelengths. An overview of earlier methods used for determination of steviol glycosides was written by Bovanová (1998). The HPLC methods describe a separation of steviol glycosides especially on columns with NH2 or C18 functional groups; the mobile phases include mostly acetonitril or methanol with water in various rations. Afandi et al (Afandi, 2013) described the optimization of conditions to obtain rebaudioside A from stevia leaves using Soxhlet extractor with subsequent HPLC determination on C18 column with mobile phase acetonitrile/water 80/20 (v/v). Bovanová et al. (Bovanová, 1998) published the preparation of liquid samples by SPE prior to HPLC analysis.</p>
<p>A new technique based on hydrophilic interactions in liquid chromatography (HILIC) is currently used to separate very polar and hydrophilic compounds. Alpert (Alpert, 1990) suggested term “chromatography of hydrophilic interactions” (HILIC) for chromatography on columns typical for systems with normal phases, which however are using water-organic mobile phases like the RP systems. The word “hydrophilic” characterizes the affinity to water added to mobile phases in HILIC separations on polar columns. The HILIC technique was first used for separation of saccharides, amino acids and peptides (Strege,1998). However, the number of newly developed columns for HILIC and applications for separations in the area of environment, foods, natural compounds, synthetic drugs and nonionic surfactants has grown very quickly in the last years. An important benefit of HILIC separations is higher sensitivity of LC-MS analyses because of improved electrospray ionization (ESI) in mobile phases with high content of acetonitrile (Brown at al., 2002; Jandera, 2011).</p>
<p>This principle can be used for HILIC separation of steviol glycosides with UV detection (Thermo, 2012). Compared to UV detection, the use of ELS detector (evaporative light scattering) provides improvement of the limits of quantification and detection. The disadvantage of this detector is the necessity to use volatile mobile phase. </p>
<p>Though the relevant literature contains many methods for determination of rebaudioside A and other steviol glycosides, only a few food laboratories use this method routinely. This is reflected in the cost of this analysis. This study therefore aimed at setting up an analytical method for the determination of rebaudioside A (Fig. 1) in mixed beer and lemonade using an available HPLC technique with UV detection and HILIC separation principle.</p>
<p>The structure of rebaudioside A with prevailing –OH groups determines its high polarity. The probable value of logarithm of distribution coefficient between water and octanol (log P) is – 3.12. Values lower than 1 characterize the polar compounds (author’s comment, see the Dictionary of Food Compounds on CD-ROM, 2004).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_1_Struktura_rebaudiosidu_A_38e60bf9ca/Obr.-1-Struktura-rebaudiosidu-A.png" alt="Fig. 1 Structure of rebaudioside A"/></p>
<h3><strong>2 MATERIAL AND METHODS</strong></h3>
<h5><strong>2.1. Chemicals and solvents</strong></h5>
<ul>
<li>Rebaudioside A, HPLC standard &gt; 96% (Sigma – Aldrich)</li>
<li>Deionized water, content of organic compounds &lt; 4 ppb (Millipore, USA)</li>
<li>Acetonitrile, Chromasolv, gradient grade, for HPLC &gt; 99.9%, Sigma Aldrich</li>
<li>Methanol, Chromasolv, gradient grade, for HPLC &gt; 99.9%, Sigma Aldrich</li>
<li>Syringe cellulosic filters 0.2 μm porosity (SISw, Prague).</li>
</ul>
<h5><strong>2.2. Sample preparation</strong></h5>
<p>Samples of lemonades are only pre-purified before analysis in view of the higher concentration of rebaudioside A. Samples of mixed beer, with much lower content of the sweetener, must be pre-concentrated so that the measured values will fi t into the range of the calibration curve of rebaudioside A. The extraction columns C18, E 1000 mg/6 ml (Strata, Phenomenex, USA) were used for purification and pre-concentration. The extraction column was first conditioned step by step with10 ml of methanol, 10 ml of deionized water and after that a sample of lemonade (5 ml) or mixed beer (25 ml) was applied on the column. The sample captured on the adsorbent was washed (10 ml of 20% acetonitrile in deionized water) and eluted into 5 ml of the mixture of acetonitrile – methanol in a 1:1 (v/v) ratio. The purified sample was loaded on the column after filtration on cellulose filter.</p>
<h5><strong>2.3. Preparation of calibration solutions</strong></h5>
<p>The range of the calibration curve was chosen to comply with determination of rebaudioside A in both lemonades and in mixed beers with concentration lower than 1 mg/l (these samples were preconcentrated). The calibration solutions were prepared by dissolving a standard in mobile phase. The stock solution of rebaudioside A was prepared by weighing 100 mg of rebaudioside A standard with accuracy ± 1 mg and its dissolution in 100 ml in graduated flask. </p>
<p>The other calibration levels of analyte, i.e. 1, 5, 10, 20 and 50 mg/100 ml were prepared by dissolving stock solution in appropriate volumes of mobile phase.</p>
<h5><strong>2.4. HPLC determination</strong></h5>
<p>HPLC with UV detection at 210 nm on Dionex Ultimate 3000 liquid chromatograph (Thermo Scientific) with Chromeleon 7 control and processing software was used for determination of rebaudioside A. The column Acclaim Mixed – Mode WAX 1 (2.1 x 150 mm; 5 μm; Thermo Scientific) in HILIC mode was used for separation of polar rebaudioside A from other compounds present in beer and lemonade matrices. The mobile phase consisted of acetonitrile and 10 mM ammonium formate at pH 3.00 ± 0.05 in a ratio of 80/20 (v/v) in isocratic mode. The flow rate of mobile phase was 0.5 ml/min, the column was temperature-controlled at 40 °C, and injection volume was 5 μl.</p>
<h3><strong>3 RESULTS AND DISCUSSION</strong></h3>
<p>The method was optimized for mixed beer and lemonade matrices, and its validation involved verification of linearity, repeatability and recovery of the method.</p>
<h5><strong>3.1 Selectivity</strong></h5>
<p>As follows from Fig. 2, the elution zone of rebaudioside A is distinctly separated from other polar analytes even for its very low concentration in the sample. The last minority component elutes at 7.04 min while the elution zone of rebaudioside A is at 7.95 min. The total separation of rebaudioside A from other components complies with the requirements for a good selectivity of the method. This is given by the use of HILIC mode which has a markedly better selectivity to polar compounds and thus to rebaudioside A than the usual non-polar columns. Another advantage of the HILIC mode is a higher sensitivity; it is 10 – 1000-fold higher for hydrophilic analytes than reverse mode techniques.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_2_Chromatogram_separace_vzorku_piva_obsahujici_rebaudiosid_A_14e5fe876d/Obr.-2-Chromatogram-separace-vzorku-piva-obsahujici-rebaudiosid-A.png" alt="Fig. 2 Chromatogram of separation of beer sample containing rebaudioside A. Column Acclaim Mixed – Mode WAX 1 (2.1 x 150 mm; 5 μm), mobile phase acetonitrile and 10 mM ammonium formate 80/20 (v/v) in isocratic mode. Flow rate of mobile phase 0.5 ml/min, temperature of column 40 °C, injection volume 5 μl. The retention time of rebaudioside A is 9.75 min"/></p>
<h5><strong>3.2 Linearity</strong></h5>
<p>The linearity was verified in a calibration range of 1-50 mg/l where the detector response of rebaudioside A has clearly a linear dependence. It is evident from table 1 that the concentrations of rebaudioside A in the original mixed beer appear in the close proximity of the lower point of the calibration curve.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_1_Opakovatelnost_metody_ca557696e5/Tab.-1-Opakovatelnost-metody.png" alt="Table 1 Repeatability of the method"/></p>
<h5><strong>3.3 Repeatability and recovery of the method</strong></h5>
<p>A Cola-type lemonade and mixed beer flavored with fruit extract, both with the stated content of rebaudioside A, were used for determination of repeatability and recovery of the method. The concentration of the monitored analyte was determined for both samples. Each sample was extracted on a SPE column and subsequently analyzed on a chromatographic column. The determined content of rebaudioside A in each sample and repeatability (expressed as a limit of repeatability) are given in Table 1. The permissible difference between two parallel determinations (limit of repeatability) in both the beer and the lemonade is 0.42 mg/100 ml, which attests to the good precision of the method.</p>
<p>The repeatability of the method was determined using standard addition of rebaudioside A to samples of mixed beer and lemonade. Two distinct concentrations of the standard were prepared to eliminate mistakes in the dosage of different volumes of standard added into the beer and the lemonade. A solution having the concentration of 1000 mg/100 ml water was prepared for spiking of lemonade while a solution of 100 mg/100 ml water was prepared for spiking beer. The standard was added into 100 ml volumetric flasks to achieve two different concentrations. With both matrices, an adequate volume of the standard was filled up to the graduation mark.</p>
<p>The dosage of 1 ml or 2 ml of standard solution into lemonade caused an increase of the analyte concentration to about 10 resp. 20 mg/100 ml. The dosage of 2 ml resp. 4 ml of standard solution into beer signified the increasing the concentration of analyte by about 2 or 4 mg/100 ml. Every concentration level was determined twice. The results are given in Table 2. The recovery of the method for lemonades and mixed beer ranges in the interval from 101.9% to 102.8% and from 90.2% to 93.1%, respectively. This fact is apparently caused by larger matrix effect in beer and by pre-concentration of beer before analysis. Due to the solubility of the analyte in alcohol its partial leak occurs during of sorption and washing of SPE column. </p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_2_Vyteznost_metody_0c8925b9c2/Tab.-2-Vyteznost-metody.png" alt="Table 2 Recovery of the method"/></p>
<p>The HILIC technique permits the separation of rebaudioside A from other polar compounds also in the very complicated beer matrix, which would not be possible using conventional polar column due to the high polarity of rebaudioside A. The elimination of co-elution of rebaudioside A with other polar compounds assures the accuracy of determination without overrating of the results. All other analytical parameters are in the agreement with the results of other published methods with UV detection in the range of short wavelengths.</p>
<h3><strong>4 CONCLUSION</strong></h3>
<p>The newly developed method to determination of rebaudioside A shows satisfactory precision (repeatability 0.42 mg/100 ml) and accuracy (recovery 90–100%). The solubility of rebaudioside A in alcohol explains the equilibrium shift on extraction column, and thus also a lower recovery from beer in comparison to non-alcoholic lemonade. The concentration of rebaudioside A in mixed beer (or a beer-based beverage) and lemonade after its treatment on the SPE column complies with a linear calibration relationship in the range from 1 to 50 mg/100 ml of the analyte.</p>


Determination of Rebaudioside A in Mixed beer, Beer-based Beverages and Lemonades

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Vědecký recenzovaný online časopis Kvasný průmysl (eISSN 2570-8619), vychází jako samostatné periodikum v angličtině. Od ročníku 2015 je obsah indexovaný databází Web of Science (edice ESCI), dále je indexován v databázích CAS, CAB Abstracts, DOAJ.
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